Analyte sensor

ABSTRACT

In one embodiment, a sensor to measure the presence of an analyte is disclosed. The sensor includes a working conductor with an electrode reactive surface. The sensor further includes a first reactive chemistry that is responsive to a first analyte and is in direct contact with the electrode reactive surface. The first reactive chemistry includes an enzyme, a first transport material, and an entrappable cofactor that includes a cofactor for the enzyme coupled to an anchor molecule. The sensor further includes a second transport material that enables diffusion of the first analyte to the first reactive chemistry.

RELATED APPLICATION

This application claims the benefit of U.S. provisional applicationnumber 63/332,645 filed on Apr. 19, 2022. The application listed aboveis hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention is generally directed to devices and methods thatperform in vivo monitoring of an analyte or analytes such as, but notlimited glucose, lactate or ketones. In particular, the devices andmethods are for electrochemical sensors that provide informationregarding the presence or amount of an analyte or analytes within asubject.

BACKGROUND OF THE INVENTION

Diabetes is a growing healthcare crisis, affecting nearly 30 millionpeople in the United States. Approximately 10 percent of those affectedrequire intensive glucose and insulin management. In hospital patients,hypoglycemia in both diabetic and non-diabetic patients is associatedwith increased cost and short- and long-term mortality.

Diabetic ketoacidosis (DKA) is a serious complication of diabetes.Diabetic ketoacidosis most often occurs in those with type 1 diabetesthough it can also occur in those with other types of diabetes. DKAtypically occurs when high levels of blood acids called ketones areproduced. The condition develops is associated with diabetes because itis linked to the lack of insulin production. Without enough insulin, thebody switches to burning fatty acids, which results in production ofacidic ketone bodies.

Accordingly, it would be highly advantageous to enable real-time in-vivodetection and measurement of ketone bodies. The claimed invention seeksto address many issues associated with detecting and measuring ketonebodies or ketones.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, a sensor to measure the presence of an analyte isdisclosed. The sensor includes a working conductor with an electrodereactive surface. The sensor further includes a first reactive chemistrythat is responsive to a first analyte and is in direct contact with theelectrode reactive surface. The first reactive chemistry includes anenzyme, a first transport material, and an entrappable cofactor thatincludes a cofactor for the enzyme being coupled to an anchor molecule.The sensor further includes a second transport material that enablesdiffusion of the first analyte to the first reactive chemistry.e.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings that illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A includes exemplary views of a first side and a second side of asensor, in accordance with embodiments of the present invention

FIG. 1B includes exemplary views of a first side and second side ofanother embodiment of the sensor, in accordance with embodiments of thepresent invention.

FIGS. 2A and 2B are exemplary illustrations of sensors that areconfigured with various combinations of single or multiple workingelectrodes, combined with single or multiple CR electrodes, or aseparate counter electrode and reference electrode, in accordance withvarious embodiments of the present invention.

FIG. 3 is an illustration showing an exemplary process that enables orcreates the entrappable cofactor that is mixed or blended or combinedinto the reactive chemistry, in accordance with embodiments of thepresent invention.

FIG. 4 is an exemplary illustration of the first reactive chemistry inaccordance with embodiments of the present invention.

FIG. 5 is an exemplary illustration of optionally incorporating anelectrically conductive element into the first reactive chemistry, inaccordance with embodiments of the present invention.

FIGS. 6A and 6B are exemplary illustrations of alternative embodimentsthat incorporate additional materials, in accordance with embodiments ofthe present invention.

FIG. 7 is exemplary calibration data generated using a sensor configuredas discussed above, in accordance with embodiments of the presentinvention.

DETAILED DESCRIPTION

Presented below are embodiments of an electrode within a sensor that isintended to enable continuous real-time in-vivo electrochemical sensingof an analyte or molecule of interest within a subject. The in-vivomeasurement within a subject is typically performed in tissue such as,but not limited to subcutaneous tissue. However, various embodiments canbe inserted into the vasculature, musculature or organ tissue. Thesensor may include a working electrode along with a counter electrodeand a reference electrode. Alternatively, many embodiments utilize aworking electrode in conjunction with a pseudo-reference electrode,alternatively referred to as a combined counter-reference electrode.

Embodiments of the sensor can be configured to measure analytes such aslactate, ketones, glucose and the like. Furthermore, while someembodiments may be configured to measure a single or individual analyte,other embodiments can be configured to measure multiple analytesincluding various combinations of at least two or more molecules ofinterest such as lactate, ketone, glucose, oxygen, reactive oxygen andthe like. In still other embodiments, the sensors may be configured withinfusion sets to enable sensing of a single or multiple molecule ofinterest while also enabling delivery of an infusate from a single pointof entry.

In many embodiments electrochemical detection of the desired analyte isaccomplished using an enzymatic reaction. In many embodiments presentedbelow, an enzyme or enzymes are selected from the dehydrogenase family.Non-limiting, exemplary dehydrogenase enzymes include glucosedehydrogenase to measure glucose and 3-hydroxybutyrate dehydrogenase(3HBDH) to measure ketones. In other embodiments, especially thosemeasuring multiple analytes, it may be possible to use alternate enzymessuch as, oxidase based enzymes like glucose oxidase or lactate oxidasein addition to dehydrogenase base enzymes. In many embodiments, theenzyme is immobilized or trapped within a reactive chemistry. In someembodiments, the reactive chemistry includes a cofactor. The inclusionof the cofactor in sufficient quantity can improve the ability tomeasure analytes having generally low endogenous concentrations ofcofactor by ensuring the kinetics of an enzyme and associated cofactorare limited by mass transfer of the molecule of interest. Particularly,inclusion of the cofactor can improve sensor properties such as, but notlimited to, linearity of the sensor response to increasingconcentrations of the analyte and increased duration of the sensorlifespan.

When dehydrogenase enzymes are used within the reactive chemistry, itmay be beneficial to include or provide a cofactor. The inclusion of acofactor within the sensor may be desirable because endogenousconcentrations of the cofactor may be low or suboptimal for variousdesign parameters of an electrochemical sensor. Accordingly, techniquesor methods to incorporate or include a desired exogenous cofactor withinthe sensor may lead to improvements of in vivo sensor performancecharacteristics such as, but not limited to, sensitivity, linearity, andstability over time.

In many embodiments, it is desirable to locate an exogenous cofactor inclose proximity to the corresponding enzyme. However, because thecofactor is often a much smaller molecule than the enzyme, it can bedifficult to prevent the cofactor from migrating or moving away from theenzyme. To prevent migration or movement of the cofactor within thereactive chemistry, in many embodiments it is preferred to minimizediffusion, entrap or immobilize the cofactor in a location relativelyclose to the enzyme. Collocating sufficient amounts or quantities of thecofactor in close proximity to sufficient quantities of the enzymeenables the kinetics of the immobilized or entrapped enzyme and cofactorto be limited by mass transfer of the analyte or molecule of interestinto the combined enzyme and cofactor structure. In some embodiments,retention or entrapment of the cofactor is accomplished by attaching thecofactor to an anchor molecule to create an entrappable cofactor. Inthese embodiments, the anchor molecule is a molecule having physicalproperties that minimize, reduce, or prevent migration or movement ofthe entrappable cofactor within the reactive chemistry that includes theenzyme. For example, an in-vivo sensor designed to continuously monitoror measure ketones, the reactive chemistry may utilize 3HBDH as theenzyme and may benefit from having exogenous cofactor NAD+ or NAD(P)+entrapped, collocated, or immobilized in close proximity to the 3HBDHenzyme.

Disclosed below are exemplary, non-limiting embodiments of a sensorhaving a reactive chemistry that includes one or more enzymes along withat least a cofactor associated with at least one of the enzymes beingcollocated, entrapped, anchored or immobilized in close proximity to theenzyme. In many of the illustrations and much of the accompanyingdescription, the exemplary non-limiting cofactor is NAD+ or NAD(P)+because it is a cofactor commonly associated with the 3HBDH enzyme.However, other embodiments may use different cofactors that are eitherthe natural cofactor, derivative of the natural cofactor, or asemi-synthetic or synthetic analogue of the natural cofactor that arecompatible with the enzyme associated with detection or measurement ofthe molecule or analyte of interest. For example, in embodiments wherethe enzyme is 3HBDH, the natural cofactor NAD+. A derivative of thenatural cofactor is NAD+ with a chemical modification or modifications,such as, but not limited to hexylamine NAD+. Additionally, asemi-synthetic or synthetic analogue of the natural cofactor that isenzymatically active. Additionally, the various embodiments discussedbelow should not be viewed as discrete embodiments. Rather, it isintended that various elements or components of the various embodimentsare intended to be combined with elements, features or components of theother embodiments.

While embodiments and examples may be related to particular figures thescope of the disclosure and claims should not be construed to be limitedto the explicit embodiments discussed. Rather it should be recognizedthat various combinations of features, elements and components can beinterchanged, combined and even subtracted to enable other embodimentscapable of continuous, real-time detection and measurement of an analyteor multiple analytes indicative of various metabolic conditions orgeneral physiological health.

FIG. 1A includes exemplary views of a first side 102 a and a second side102 b of a sensor 100, in accordance with embodiments of the presentinvention. FIG. 1A further includes exemplary cross-sections A-A-1 andA-A-2 of the sensor 100. As illustrated the sensor 100 includes at leasttwo electrical traces. One of the electrical traces is configured to bea working trace 104 a that supports working electrodes (WE) 106. Asecond electrical trace is configured to be a counter/reference (CR)trace 104 b that supports a combined CR electrode (CRE) 108. Throughoutthis disclosure, the term working trace 104 a may be interchanged withthe term working conductor, working electrode conductor, or workingelectrode trace. Similarly, the term CR trace 104 b may be interchangedwith the term CR conductor, CR electrode trace, or CRE trace. The use oftwo electrical traces to support electrochemical sensing using a twoelectrode system should not be construed as limiting. In otherembodiments, a three electrode system may be used where each of theworking electrode, counter electrode and reference electrode are formedon their own electrical trace or conductor. With some embodiments it maybe preferable to use two electrodes rather than three electrodes due tothe decreased size of the sensor 100. However, in other embodiments itmay be preferable to use a three electrode system in order to improveaspects of sensor performance such as sensitivity or linearity overtime.

In FIG. 1A the WE 106 is illustrated as a plurality or array ofapertures or openings, formed on the working trace 104 a. The number ofapertures or openings within the WE 106 array is intended to beexemplary and should not be construed as limiting. In variousembodiments various configurations having more or fewer openings may beused to tune sensor performance based on the molecule being detected orsensed. Likewise, the illustration of the CRE 108 as a monolithicopening is intended to be illustrative rather than limiting. In otherembodiments the CRE 108 may be formed from multiple openings on the CRtrace 104 b. Moreover, the ratio of area between the exposed WE 106 andCR electrode 108 provided in the illustration is intended to beexemplary rather than limiting.

In FIG. 1A both the WE 106 and the CRE 108 are exposed on the first side102 a. In preferred embodiments, the WE trace 104 a and the CR electrodetrace 104 b are formed from a contiguous piece of electrical conductor,such as, but not limited to, stainless steel, silver, gold, platinum, orthe like that is patterned to produce the working trace 104 a and the CRtrace 104 b. In other embodiments other techniques such as deposition,machining, etching and the like can be used to form or place one or moreof the working trace 104 a and the CR electrode trace 104 b. The WE 106and CRE 108 are formed toward a distal end 110 of the sensor 100. Thoughnot included in the illustration, it should be understood that the WE104 a and the CR electrode trace 104 b include contact pads formedcloser to a proximal end 112 that enable the sensor 100 to be connectedto electronic components that power and operate the sensor 100.

Cross-sections A-A(1) and A-A(2) are exemplary illustrations ofembodiments of the sensor 100, in accordance with various embodiments ofthe present invention. Common elements shared between the cross-sectionsA-A(1) and A-A(2) include an insulation 114 that is coupled to the WEtrace 104 a and the CR trace 104 b. In embodiments where the WE 106 andCRE 108 are formed on the first side 102 a, the second side 102 billustrated in FIG. 1A is the insulation 114. In many embodiments, theinsulation 114 is an insulator such as, but not limited to anon-conductive film like polyimide. In other embodiments, differentelectrical insulators such as a solder mask may be used as theinsulation 114. The specific embodiments discussed are intended to beexemplary and other non-conductive insulator materials may be used forinsulation 114.

Insulation 118 covers working trace 104 a and the CR trace 104 b and theinsulation has openings, windows, or apertures that expose a portion ofthe respective trace that enables formation of the WE 106 or the CRE108. In many embodiments insulation 118 is selected from electricalinsulators that can be applied over the respective electrode traces.Exemplary insulation 118 can include, but are not limited tonon-conductive films like polyimide that are coupled to the electrodetraces with adhesives. In other embodiments insulation 118 isselectively applied via spin coating, spraying, or alternate forms ofdepositing or placing an electrical insulator over the respectiveelectrodes. In many embodiments, the opening or apertures within theinsulation 118 that expose a portion of the underlying respectiveelectrical trace are made using techniques such as, but not limited to,laser ablation, mechanical cutting, masking, etching or the like. Thetechniques to create the apertures are not intended to be limiting. Anytechnique that can be used to selectively apply or remove the insulation118 from the respective electrical traces should be considered withinthe scope of this disclosure. For example, in some embodiments theinsulation 118 may be printed over an electrical trace leaving theapertures exposed thereby obviating the need to remove the insulation toform the aperture. As illustrated the openings in the insulation 118that expose the working conductor 104 a are circular but that should notbe construed as limiting. Rather, any shape opening or aperture withinthe insulation 118 should be considered within the scope of thisdisclosure. Similarly, the shape of the opening in the insulation 1180that exposes the CR conductor 104 b is intended to be exemplary and maybe any variety of shape.

Additional common element shared between the cross-sections A-A (1) andA-A (2) is a first reactive chemistry 122 that in many embodimentsfurther includes an enzyme 122 a, a first transport material 122 b andan entrappable cofactor 122 c. Another common element betweencross-sections A-A (1) and A-A (2) is a second transport material 120.In cross-section A-A (1) the second transport material 120 is appliedover the insulation 118 and the working conductor 104 a and the CRconductor 104 b. In preferred embodiments the second transport material120 is a hydrogel that extends from an edge 126 a to an edge 126 b. Thepurpose of the second transport material 120 is to enable unencumberedmovement, diffusion or transport of molecules within fluid surroundingthe sensor 100. Accordingly, after a hydration period, the secondtransport material 120 is intended to establish molecular concentrationswithin the second transport material 120 that are substantiallyidentical to those within the fluid surrounding the sensor (other thanthose molecules that are intended to react within the sensor 100).

A first reactive chemistry 122 is applied over the second transportmaterial 120 in substantial alignment with the exposed WE trace 104 a tocreate a working electrode 106. The first reactive chemistry includesthe enzyme 122 a, the first transport material 122 b and the entrappablecofactor 122 c. In many embodiments, both the enzyme 122 a and theentrappable cofactor are selected based on a molecule intended to bemeasured or detected by the working electrode 106. For example, inembodiments where ketones are the molecule of interest, the enzyme 122 aincluded within the first reactive chemistry 122 may include adehydrogenase based enzyme such as 3HBDH. The use of 3HBDH should not beconstrued as limiting as other embodiments may require the use ofmolecule specific enzymes. Non-limiting examples of other enzymes 122 ainclude, but are not limited to detection or measuring of analytes ormolecules of interest like glucose or lactate that are capable of beingdetected or measured with oxidase enzymes such as glucose or lactateoxidase or dehydrogenase based enzymes such as glucose or lactatedehydrogenase. In embodiments utilizing dehydrogenase based enzymes, itmay be advantageous or necessary to include supplemental or additionalcofactor to enable electrochemical sensing of the molecule of interest.

Accordingly, in many embodiments, the reactive chemistry 122 furtherincludes an entrappable cofactor 122 c along with a first transportmaterial 122 b. In many embodiments, the first transport material 122 bis a polymer that is crosslinked. Crosslinking of the first transportmaterial defines a porosity that entraps or ensnares the enzyme 122 aand the entrappable cofactor 122 c. In many embodiments, the firsttransport material 122 b is selected from a family of hydrogels havingsimilar properties to those of the second transport material. Inpreferred embodiments, when the reactive chemistry 122, a mixture orcompound including the enzyme 122 a, the first transport material 122 band the entrappable cofactor 122 c is crosslinked or cured, the firsttransport material 122 b creates a porous matrix that minimizes orprevents migration or movement of both the enzyme 122 a and theentrappable cofactor 122 c within or throughout the first transportmaterials 122 b.

In preferred embodiments, the porosity of the first transport material122 b is selected to restrict or minimize migration or movement of theenzyme 122 a and the entrappable cofactor 122 c while allowingunrestricted or minimally restricted migration or movement of theanalyte of interest, endogenous cofactor, and other endogenous moleculesor compounds found within interstitial fluid within a subject. Retainingthe entrappable cofactor 122 c within the first reactive chemistry 122supplements or enhances endogenous cofactor within a subject that mayexist in generally low concentrations. Because endogenous cofactor inlow concentrations may be quickly depleted within the sensor, theinclusion of the entrappable cofactor 122 c is intended to improve orenhance continuous sensor performance over an expected sensor life thatcan be measured in multiple hours, days or weeks.

The porosity of the first transport material 122 b is intended to freelyenable diffusion or transport of endogenous cofactor which necessitatesthe purposeful modification or tuning of the entrappable cofactor 122 cto achieve a desired diffusivity of the entrappable cofactor within thefirst transport material 122 b. Specifically, the entrappable cofactor122 b is modified to minimize or prevent diffusion of the entrappablecofactor 122 b within or throughout the first transport material 122 b.With diffusion within the first transport material minimized, theentrappable cofactor 122 b is retained in close proximity to the enzyme122 a while still enabling diffusion or transmission fluids containingendogenous cofactor, the analyte of interest and other molecules orcompounds found in fluid surrounding the sensor within and throughoutthe first transport material 122 b. The doping or addition ofentrappable cofactor 122 b that cannot migrate or diffuse away from theenzyme 122 a improves sensor performance by compensating for anyshortage of endogenous cofactor within a subject.

In many embodiments the shape of the first reactive chemistry 122mirrors, or is at least similar to the shape of the aperture in theinsulation 118. However, it should be noted that the first reactivechemistry 122 and a portion of the exposed WE trace may be the sameshape or different shapes. Similarly, the exposed WE trace and the firstreactive chemistry 122 may be nominally identical in size, or one may belarger or smaller than the other. In many embodiments, the firstreactive chemistry 122 is sized and positioned such that if the firstreactive chemistry 122 were projected upon the exposed working trace 104a, it would overlap or at least cast a shadow over at least some of theexposed working trace. The overlap or shadow cast by the first reactivechemistry 122 ensures that byproducts of the reaction between themolecule of interest and the first reactive chemistry 122 have asubstantially direct path from the first reactive chemistry 122, throughthe second transport material 120, to the exposed working trace.

A third transport material 124 is optional and may be applied over boththe first reactive chemistry 122 and the second transport material 120.Though illustrated as being applied from edge 126 a to edge 126 b, invarious embodiments the third transport material 124 may be applied suchthat it does not extend to either, or both edges 126 a and 126 b. Insome embodiments the third transport material 124 is impervious to themolecule of interest. In other embodiments, the third transport material124 enables fluid surrounding an implanted sensor containing the analyteof interest, the cofactor and other molecules and compounds within thefluid to freely migrate or move throughout the third transport material124. In many embodiments, the third transport material 124 is similar oridentical to either or both of the first transport material and thesecond transport material.

Cross-section A-A (2) differs from cross-section A-A (1) because withA-A (2), the first reactive chemistry 122 is applied to the exposed WEtrace 104 a. In cross-section A-A (1), the first reactive chemistry 122is not in direct contact with the WE trace 104 a. Rather, withcross-section A-A (1), the first reactive chemistry is separated fromthe WE trace 104 a by the second transport material 120. Incross-section A-A(1), an optional third transport material 124 may beapplied over both the second transport material 120 and the firstreactive chemistry 122. As illustrated in cross-section A-A (1), thefirst reactive chemistry 122 is sandwiched or encapsulated between thesecond transport material 120 and the third transport material 124. Inmany of these embodiments, the third transport material is intended topromote biocompatibility of the sensor by preventing or minimizing thelikelihood that body fluid around an implanted sensor interacts directlywith the first reactive chemistry 122. Additionally, in cross-sectionA-A (2), optional third transport material 124 is applied over thesecond transport material 120. In some embodiments where the secondtransport material 120 is applied directly over and in contact with thefirst reactive chemistry 122, the second transport material 120 operatesas a biocompatibility layer. Specifically, in these embodiments thesecond transport material 120 operates as a barrier to minimize thelikelihood of interstitial fluid surrounding an implanted sensor fromdirectly interacting with the reactive chemistry 122.

FIG. 1B includes exemplary views of a first side 102 a and second side102 b of another embodiment of the sensor 100, in accordance withembodiments of the present invention. FIG. 1B further includes exemplarycross-sections B-B (1) and B-B (2) of the sensor 100. As illustrated inFIG. 1B, the working trace 104 a is formed on the first side 102 a andthe CR trace 104 b is formed on the second side 102 b. Similar to FIG.1A, the use of a two electrode system is exemplary rather than limitingas other embodiments can use a three electrode system having separatetraces for each of the working, counter and reference electrodes. Insuch embodiments, splitting the three electrodes across the first side102 a and second side 102 b results in various permutations orcombinations that should be considered within the scope of thisdisclosure despite the focus being on the exemplary two electrodesystem. It may be preferable to use an embodiment similar to thatillustrated in FIG. 1B to reduce the overall width of the distal end110, despite the increase in thickness of the edges 126 a and 126 b.

In FIG. 1B, the working electrode 106 is illustrated as a plurality ofopenings, or an array of openings or apertures, formed on the workingtrace 104 a while the CR electrode 108 is illustrated as a singleopening formed on the CR trace 104 b. Similar to the embodimentillustrated in FIG. 1A, the CR electrode 108 is a single relativelylarge opening on the CR trace 104 b that should not be construed aslimiting. Rather the CR electrode 108 may also be configured as an arraymade up of multiple openings on the CR trace 104 b. The exemplary numberof openings within the WE 106 array should not be construed as limitingas other embodiments may include an WE 106 with an array with fewer oradditional openings. Changing the number of openings may be used to tunesensor performance based on the relative concentration of the moleculebeing sensed. For example if there is a relatively low concentration ofthe molecule being sensed or detected, it may be advantageous toincrease the number of openings within the WE 106 array. Alternatively,in embodiments where the concentration of the molecule being sensed isrelatively high, decreasing the number of openings within the WE 106array may result in satisfactory sensor performance while reducingmanufacturing cost and complexity.

In addition to the number of openings within the array of workingelectrodes, sensor performance may be further tuned or enhanced bychanging the surface area exposed within each of the openings viaremoval of more or less insulation 118. Larger openings may enablegreater quantities of first reactive chemistry (e.g., enzyme) to be usedthereby potentially generating higher electrical currents. Conversely,smaller openings may enable smaller quantities of a first reactivechemistry 122 (e.g., enzyme) to be used to generate sufficientelectrical current to enable electrochemical sensing of the molecule ofinterest.

With the WE 106 being formed on the first side 102 a and the CRelectrode 108 being formed on the second side 102 b, the respectiveelectrical traces are electrically isolated from each other withinsulation 114 (cross-sections B-B (1) and B-B (2)). In preferredembodiments, the working trace 104 a and CR trace 104 b are made from aflexible, electrically conductive material such as, but not limited tostainless steel. In alternative embodiments alternate materials otherthan stainless steel may be used to form one or more of the electricaltraces. Non-limiting examples include, but are not limited to minimallyor non-corrosive, electrically conductive and flexible materials such asgold, silver, platinum or alloys thereof. Still other materials includecarbon nano-tubes or other conductive materials with a non-corrosivecoating applied thereto. In preferred embodiments the WE trace iscreated by patterning a contiguous piece of the electrical conductor.Similarly, the CR trace may be created by patterning a separate piece ofelectrical conductor.

Cross-sections B-B (1) and B-B (2) are exemplary illustrations ofembodiments of the sensor 100, in accordance with embodiments of thepresent invention. Similar to FIG. 1A, the cross-sections includeinsulation 114 and insulation 118 that are coupled to both working trace104 a and CR trace 104 b. In some embodiments insulation 114 is a singlelayer of insulating material that is coupled to the working trace 104 aand the CR trace 104 b using adhesives. In other embodiments, insulation114 is made of multiple non-conductive insulators laminated togetherthat are coupled to the working trace 104 a and the CR trace 104 b.Non-limiting examples that can be used for insulation 114 and insulation118 include polyamide, solder mask and similar materials. Non-limitingexamples of the adhesives that can be used to couple the respectivetraces 104 a and 104 b to the insulation 114 and/or insulation 118include acrylics or epoxies.

The WE 106 and CR electrode 108 are formed by creating apertures oropening within the insulation 118 that covers the respective electricaltrace. The opening in the insulation 118 exposes a portion of either theWE trace 104 a or the CR electrode trace 104 b. The openings orapertures within the insulation 118 can be formed using laser ablation,mechanical cutting, masking and etching or any other technique toselectively remove a portion of the insulation 118 to expose therespective underlying electrical trace. Alternatively, in otherembodiments rather than removing insulation 118 to expose an underlyingelectrical trace, the insulation 118 may be applied already havingapertures formed in the insulation 118. As discussed regarding FIG. 1A,the shape of the apertures or openings in FIG. 1B are exemplary andshould not be construed as limiting. Various embodiments may haveapertures of various sizes, shapes and areas depending on theperformance characteristics of the sensor 100.

Cross-sections B-B (1) and B-B(2) have the second transport material 120applied over the insulation 118 and the exposed portion of the CR trace104 b. Cross-section B-B(1) also has the second transport material 120applied over the insulation 118 and the exposed portion of the WE trace104 a. Cross-section B-B (1) has the first reactive chemistry 122located over, but separated from the WE trace 104 a, by the secondtransport material 120. Cross-section B-B(2) has the first reactivechemistry selectively applied over a portion of the insulation 118 andthe WE trace 104 a. It should be noted that the embodiments illustratedare exemplary and alternative embodiments that modify aspects of theexemplary embodiments should be considered within the scope of thedisclosure. For example, embodiments similar to A-A(2) of FIGS. 1A andB-B(2) of FIG. 1B may partially fill the aperture or window of theinsulation 118 thereby leaving a portion of the working conductor 104 aexposed to be subsequently covered by first transport material 120.Similarly, in FIGS. A-A (1) and B-B (1) the reactive chemistry 122overlap portions of the insulation 118 rather than being similar in sizeto the opening or aperture in the insulation 118.

In both FIGS. B-B (1) and B-B (2), a third transport material 124 may beoptionally applied. In FIGS. B-B (1), the third transport material 124is applied over both the first transport material 120 and the reactivechemistry 122. In FIGS. B-B (2), the third transport material 124 may beoptionally applied over the second transport material 120. In variousembodiments, the optional third transport material 124 may be appliedacross the entire sensor 100 from edge 126 a to edge 126 b. In otherembodiments, the third transport material 124 does not extend from edge126 a to edge 126 b.

In some embodiments the third transport material 124 is similar oridentical to the second transport material 120. In other embodiments,the third transport material 124 is selected based on other propertiessuch as, but not limited to, its ability to enable or preventtransmission of one or more molecules or compounds associated with theelectrochemical reaction between the analyte of interest and thereactive chemistry.

FIGS. 2A and 2B are exemplary illustrations of sensors 100 that areconfigured with various combinations of single or multiple workingelectrodes, combined with single or multiple CR electrodes, or aseparate counter electrode and reference electrode, in accordance withvarious embodiments of the present invention. FIG. 2A is illustrative ofa sensor 100 having a first trace 200 and a second trace 202 on thefirst side 102 a a third trace 208 on the second side 102 b. In oneembodiment, the different traces can be configured to have two workingelectrodes and a combined counter/reference electrode that is sharedbetween the two working electrodes. In other embodiments, the differenttraces can be configured to have a single working electrode, a discretecounter electrode, and a discrete reference electrode.

FIG. 2B is illustrative of a sensor 100 having a first trace 200 and asecond trace 202 on the first side 102 a. Additionally, in FIG. 2B, thesensor includes a third trace 208 and a fourth trace on the second side102 b. Accordingly, in some embodiments the first trace 200 may beconfigured to be a CRE and the second trace 202 is configured to be afirst WE for a first molecule of interest. This leaves the third traceto be another CRE and the fourth trace to be a second WE for a secondmolecule of interest. Alternatively, two of the traces can be configuredas a first and second WE while the third trace is configured as adiscrete counter electrode and the fourth trace is configured as adiscrete reference electrode. Such an embodiment would enable detectionof two molecules of interest on different working electrodes bothsharing a counter electrode and a working electrode.

In the embodiments discussed above, any of the reactive chemistries mayincorporate or include various enzymes depending on the molecule oranalyte of interest. Various types of enzymes that may be included aspart of the reactive chemistry include, but are not limited todehydrogenase based enzymes and/or oxidase based enzymes. In embodimentsutilizing dehydrogenase based enzymes it may be desirable or preferableto incorporate a corresponding cofactor within close proximity to theenzyme. For example, if the analyte of interest is ketones, 3HBDH is thedehydrogenase based enzyme and inclusion or addition of the cofactorNAD+ or NAD(P)+ in close proximity to the 3HBDH may improve sensorperformance. Accordingly, in many embodiments, it may be preferable toentrap or retain NAD+ within the reactive chemistry. Because of therelatively low molecular weight of a cofactor relative to both theenzyme and the molecule or analyte of interest, an alternativeperspective may be to view the entrapment or retention of the cofactoras minimizing movement or at least the mobility or likelihood ofmovement of the cofactor within the reactive chemistry.

FIG. 3 is an illustration showing an exemplary process that enables orcreates the entrappable cofactor 122 c that is mixed or blended orcombined into the reactive chemistry, in accordance with embodiments ofthe present invention. The process described below is intended toattach, couple or bond a desired cofactor 300 to an anchor molecule 302to create the entrappable cofactor 122 c that is a combination of aportion of the anchor molecule 302 and the cofactor 300. The intent ofthe process is to modify or tune the diffusivity of the resultingentrappable cofactor 122 c to enable, improve or enhance its ability tobe entrapped, immobilized or contained within the reactive chemistry.The exemplary process illustrated in FIG. 3 uses a methacrylatefunctional group 302 a, a polyethylene glycol (PEG) backbone 302 b andsuccinimide group 302 c as the anchor molecule 302. The process replacesthe succinimide group 302 c of the anchor molecule 302 with the desiredcofactor 300 resulting in the entrappable cofactor 122 c.

The use of a PEG backbone as the anchor molecule 302 for the entrappablecofactor 122 c is intended to be exemplary rather than limiting. Inother embodiments other anchor molecules 302 such as, but not limited topolymers like polyethyleneimine and alginate may be used to tune thediffusivity of the entrappable cofactor within the first transportmaterial. In some embodiments tuning or modification of the diffusivityis accomplished by attaching the cofactor 300 to an anchor molecule 302that significantly increases the molecular size and/or molecular weightof the resulting entrappable cofactor 122 c. While a preferred molecularmass may be dependent upon multiple variables such as, but not limitedto, porosity of the first transport material and molecular mass of thecofactor 300, in many embodiments and exemplary range for the molecularmass of the anchor molecule 302 is between 100 and 100,000 daltons. Instill other embodiments, another exemplary range for the molecular massof the anchor molecule 302 is between 500 and 50,000 daltons. In manypreferred embodiments, the molecular mass of the anchor molecule 302falls between a range of 1,000 and 20,000 daltons. Accordingly, theentrappable cofactor 122 c that includes the cofactor 300 and anchormolecule 302 of significant size or preferred molecular mass has adecreased diffusivity (alternatively, increased entrapability) comparedto just the cofactor 300 within the porous matrix of the first transportmaterial.

In alternative embodiments, rather than relying on molecular mass orphysical size relative to porosity of the first transport material, thecofactor 300 can be attached to an anchor molecule that imparts anelectrical charge to the resulting entrappable cofactor 122 c thatpromotes entrapment, immobilization or retention of the entrappablecofactor 122 c within the reactive chemistry. In embodiments where theelectrical charge associated with the anchor molecule 302 reducesdiffusivity, electrical charge of the first transport material may bemore relevant than porosity. Alternatively, in embodiments havingelectrically charged anchor molecules 302, the first reactive chemistrymay further include a specific molecule having an electrical charge thatis opposite to the charge of the anchor molecule 302. In still otherembodiments, an anchor molecule 302 can be selected based on size and/ormolecular mass and its ability to impart an electrical charge. In theseembodiments, the remainder of elements within the first reactivechemistry (e.g., the first transport material, the enzyme or evensupplemental compounds or molecules) may be selected based on bothporosity and an electrical charge that attracts the anchor molecule)

The exemplary PEGylation process illustrated in FIG. 3 is used toreplace a succinimide group 302 c with a specific or desired cofactor300. In FIG. 3 , the desired cofactor 300 is NAD+ or NAD(P)+ but thatshould not be construed as limiting. Throughout this disclosure itshould be understood that the cofactor may be selected based on a singleor multiple requirements or preferences such as, but not limited to, anenzyme or enzymes being used, an analyte or analytes being detected orsensed, and the general availability of endogenous cofactor associatedwith either an analyte, an enzyme, or analytes or enzymes. Additionally,the inclusion of the succinimide group 302 c on the PEG backbone 302 bshould be viewed as illustrative rather than limiting. In otherembodiments, the PEG backbone 302 b can include functional groups otherthan, or in addition to the succinimide group illustrated. Additionalexemplary function groups include, but are not limited to, carboxylicacid, amine and biotin. Moreover, in still other embodiments, ratherthan replacing a single functional group, an anchor molecule may havemultiple attachment locations from one or more functional groups thatcan be replaced with cofactor, other molecules with desired properties,or other components of the reactive chemistry or sensor.

As discussed above, selection of the anchor molecule can be influencedby the desired molecular size and/or molecular weight of the resultingentrappable cofactor 122 c (i.e., the anchor molecule that includes themethacrylate functional group, the PEG backbone, and the desiredcofactor). Selection of the molecular size or weight of the PEGylatedcofactor can be used to improve entrapability of the cofactor within thefirst transport material incorporated into the reactive chemistrythereby improving overall sensor stability and/or functionality of thesensor.

FIG. 4 is an exemplary illustration of the first reactive chemistry 122in accordance with embodiments of the present invention. In FIG. 4 , theentrappable cofactor 122 c created during the process described above iscombined, mixed, or blended with an enzyme 122 a and the first transportmaterial 122 b to create a first reactive chemistry 122. Closelycollocating sufficient entrappable cofactor 122 c in close proximity tothe enzyme 122 a within the first transport material 122 b to create thefirst reactive chemistry structure enables kinetics of reactions betweenthe enzyme and cofactor to be limited by mass transfer of the moleculeof interest into the first reactive chemistry structure. In FIG. 4 , theenzyme 122 a is specified broadly as being a dehydrogenase enzyme.However, in different embodiments alternative types of enzymes may beused depending on the desired reaction and analyte or molecule ofinterest. For example, in FIG. 4 , the dehydrogenase enzyme 3HBDH wouldfunction with the entrappable cofactor 122 c including either NAD+ orNAD(P)+.

Variables that can be controlled or tuned during the creation of thefirst reactive chemistry 122 include, but are not limited to theconcentration or amount of the entrappable cofactor 122 c, the amount ofenzyme 122 a or enzyme loading or enzyme concentration, and the amountof first transport material 122 b. While there may be a preferred enzymebased on the entrappable cofactor (e.g., where the enzyme is 3HBDH thecofactor is NAD+ or NAD(P)+), in some embodiments additional enzymes maybe mixed with the entrappable cofactor resulting in a reactive chemistrythat includes multiple enzymes. Examples of multi-enzyme reactivechemistries include, but are not limited to multiple dehydrogenaseenzymes or reactive chemistries that include various combinations ofdehydrogenase enzymes and oxidase enzymes (e.g., 3HBDH andNADH-oxidase). In embodiments configured to measure ketones, exemplaryconcentrations of an enzyme like 3HBDH can vary between 1 -80 wt% withthe weight percent being highly dependent on enzyme activity. In theseembodiments, the exemplary concentration of entrappable cofactor NAD+can vary between 5 -95 wt%, where the weight percent may be mostlydetermined by the size or weight of the selected anchor molecule. Inmany of these embodiments, the first transport material can vary between1 -90 wt%, again, the weight percent being determined by the otherconstituents within the reactive chemistry and the desired physicalproperties of the reactive chemistry. It should be understood that theranges provided are intended to be illustrative of embodiments based onexemplary design parameters. Accordingly, the examples discussed aboveshould be construed as exemplary rather than limiting.

In addition to varying which enzyme or enzymes along with the respectiveconcentrations of the respective enzymes and entrappable cofactor,selection of the first transport material may also be based on one ormore of a desired degree of crosslinking, a desired cure time and adesired cure duration. Changing the degree of crosslinking via the firsttransport material can increase or decrease the mobility of theentrappable cofactor throughout the reactive chemistry. For example,assuming the exemplary entrappable cofactor remains the same, inembodiments with less crosslinking the entrappable cofactor may be ableto migrate or diffuse more easily through the reactive chemistrycompared to an embodiment where the first transport material has ahigher degree of crosslinking.

An additional consideration when selecting the first transport materialmay also be the wavelength of light necessary to cure the firsttransport material being introduced into the first reactive chemistry.Still another variable associated with cure time and/or the curewavelength includes the properties of the methacrylate functional group.In different embodiments, the selection of the methacrylate group incombination with the properties of the first transport material can beselected, tuned or optimized to achieve a desired polymerization of thefirst reactive chemistry. In many embodiments where the first transportmaterial is a hydrogel, the hydrogel may include a photoinitiator topromote curing or crosslinking. In various embodiments, a separate oradditional photoinitiator may be used to further refine or tune aspectsassociated with curing the first transport material.

FIG. 5 is an exemplary illustration of optionally incorporating anelectrically conductive element 500 into the first reactive chemistry122, in accordance with embodiments of the present invention. In FIG. 5, the left side is an exemplary illustration of the first reactivechemistry 122 described above. Specifically, the first reactivechemistry 122 includes the first transport material 122 b, the enzyme122 a (3HBDH) and the entrappable cofactor 122 c. As illustrated on theright side of FIG. 5 , the addition, incorporation, orelectropolymerization of the electrically conductive element 500, orconductive element, enables electrical contact between the electrodesurface and the first reactive chemistry 122. Moreover, thoroughlymixing, incorporating, or electropolymerizing the conductive element 500into or through the first reactive chemistry 122 enhances or improvescharge transport throughout the first reactive chemistry 122. Thus, whenthe first reactive chemistry 122 that includes a conductive element 500is in physical contact with the WE the conductive element 500 enablescharge transport from the WE throughout the first reactive chemistry 122including to both the enzyme 122 a and the entrappable cofactor 122 c.

In still other embodiments, to enable, enhance or improve electricalconductivity, the conductive element 500 may be incorporated into thevarious other transport materials associated with the sensor. Forexample, the conductive elements 500 may be optionally included in thesecond transport material to enable electrical conductivity through thesecond transport material, in embodiments where the first reactivechemistry is separated from the WE by the second transport material.

An exemplary, non-limiting conductive element includes variousembodiments of conductive polymer such aspoly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) commonlyreferred to as PEDOT-PSS. Conductive polymers like PEDOT-PSS enableelectron transport via movement of delocalized electrons along theconjugated backbone. The conjugated backbone has alternating single anddouble bonds that result in overlapping p-orbitals that create anextended π-conjugated system. Interpolymer change transport is thusenabled through movement of delocalized electron movement through theconjugated system or electron transfer. It should be noted that electrontransport using conductive polymers is different from electron transportusing redox polymers. Specifically, electron transport with redoxpolymers occurs via electron transfer between spatially andelectrostatically localized redox sites linked to a polymeric backbone.

Additional benefits of conductive polymers include the ability to modifythe chemical structure to change the physical properties that in turncan change the interactions of the conductive polymer with thesurrounding environment. For example, in some embodiments modificationsto the chemical structure of the conductive polymer enables tuning ofthe hydrophobicity of the conductive polymer. Still other modificationsto the conductive polymer enable tailoring of adhesion properties to aconducting or non-conducting material. In other embodiments, theconductive polymer may be modified in order to selectively functionalizeportions or the entirety of the conductive polymer.

Another benefit of incorporating a conductive polymer within the firstreactive chemistry are the multiple methods for polymerizing conductivepolymers from their respective monomers. For example, in someembodiments the conductive polymer may be polymerized via chemicalpolymerization where preferred chemicals act as an oxidant.Non-limiting, exemplary chemicals to enable chemical polymerizationinclude, but are not limited to metal ions, iron (III)-sulfonates, andthe like. In other embodiments, the conductive polymer relies onelectrochemical polymerization. Exemplary types of electrochemicalpolymerization includes, but are not limited to potentiostaticelectrochemical polymerization and galvanostatic electrochemicalpolymerization.

Furthermore, with conductive polymers, the physical and electrochemicalproperties of the conductive polymer can be tuned by modifyingpreparation parameters such as, but not limited to the polymerizationmethod, dopant selection and the like. In many embodiments, alternativeconductive elements may be used such as, but not limited to carbon orgraphene. In still other embodiments, the conductive element may be acombination of multiple conductive materials or elements such asconductive polymers and carbon nanotubes.

FIGS. 6A and 6B are exemplary illustrations of alternative embodimentsthat incorporate additional materials, in accordance with embodiments ofthe present invention. In FIGS. 6A and 6B additional or supplementalmaterials or compounds may be included or mixed into the first reactivechemistry 122 or alternatively, into the second transport material 120.An exemplary, non-limiting inclusion that may be optionally incorporatedinto either the first reactive chemistry 122 or the second transportmaterial 120 is NADH peroxidase. In embodiments utilizing 3HBDH as theenzyme, the inclusion of NADH peroxidase can promote conversion ofperoxide and NADH from fluid surrounding an implanted sensor into NAD+.

FIG. 6B further includes an optional surface preparation 600 that islocated on at least a portion of the exposed working trace. In someembodiments the surface preparation 600 is the application of a mediatorsuch as, but not limited to, poly-TBO. In other embodiments the surfacepreparation 600 includes the application of a surface enhancement thatincreases the surface area of the electrode surface.

FIG. 7 is exemplary calibration data generated using a sensor configuredas discussed above, in accordance with embodiments of the presentinvention. The data is a plot of electrical current versus time acquiredfrom a sensor where the analyte of interest or molecule being detectedwas ketones. The sensor that generated the data used 3HBDH as theenzyme, a hydrogel as the first transport material and NAD+ attached toa PEG backbone as the entrappable cofactor.

The embodiments discussed above are intended to disclose the inclusionof an exogenous cofactor with tunable or controlled diffusion propertieswithin an implantable in-vivo sensor to enable detection of sensing ofan analyte or molecule of interest. The inclusion of the entrappablecofactor is intended to improve aspects of sensor performance such assensitivity and duration of sensor life span by ensuring there issufficient cofactor to react with the analyte of interest and enzyme toproduce an electrochemical signal. Entrapping or minimizing diffusion ofthe cofactor within the reactive chemistry retains the cofactor in closeproximity to the enzyme while also allowing endogenous cofactor, themolecule of interest and by-products of the electrochemical reaction todiffuse throughout the sensor.

As described above, modifying diffusivity of a cofactor for enzymaticelectrochemical sensing may be accomplished using a variety oftechniques such as modifying the cofactor to be an entrappable cofactor.An entrappable cofactor has properties that encumbers, retards orminimizes diffusion of the entrappable cofactor within a matrix ormixture of materials surrounding the cofactor. One technique toaccomplish the reduced diffusivity of an entrappable cofactor is bymanipulating or tuning the properties of both the entrappable cofactorand the material being mixed or blended with the entrappable cofactor.

Creating the entrappable cofactor can include modifying a cofactor toinclude an anchor molecule. The anchor molecule augmenting theproperties of the cofactor to create an entrappable cofactor withpreferred properties such as, but not limited to, molecular size,molecular weight or electrical charge. The exemplary embodimentsdiscussed above should not be construed as limiting. In otherembodiments an entrappable cofactor can achieve a desired size, weightor electrical charge by attaching or coupling the cofactor to variouscombinations of anchor molecule, enzyme, additional cofactor, or variouscombinations thereof. Thus, while FIG. 3 illustrates a cofactor attachedto an anchor molecule, in various other embodiments, a cofactor may bebonded to an anchor molecule and enzyme may also be bonded to the sameanchor molecule to create the entrappable cofactor.

In many embodiments, additional features or elements can be included,added or substituted for some or all of the exemplary features describedabove. Alternatively, in other embodiments, fewer features or elementscan be included or removed from the exemplary features described above.In still other embodiments, where possible, combinations of elements orfeatures discussed or disclosed incongruously may be combined togetherin a single embodiment rather than discreetly or in the specificcombinations described in the exemplary description found above.Accordingly, while the description above refers to particularembodiments of the invention, it will be understood that manymodifications or combinations of the disclosed embodiments may be madewithout departing from the spirit thereof. The presently disclosedembodiments are therefore to be considered in all respects asillustrative and not restrictive.

1. A sensor to measure the presence of an analyte, comprising: a workingconductor having an electrode reactive surface; a first reactivechemistry being responsive to a first analyte, and in direct contactwith the electrode reactive surface, the first reactive chemistryincluding an enzyme, a first transport material, and an entrappablecofactor that includes a cofactor for the enzyme coupled to an anchormolecule; and a second transport material that enables flux of the firstanalyte to the first reactive chemistry.
 2. The sensor to measure thepresence of an analyte as described in claim 1, wherein the firsttransport material is a crosslinked polymer having a porosity based on alevel of crosslinking.
 3. The sensor to measure the presence of ananalyte as described in claim 2, wherein the anchor molecule and theporosity define a preferred diffusivity of the entrappable cofactorwithin the first transport material.
 4. The sensor to measure thepresence of an analyte as described in claim 3, wherein the tunablediffusivity of the entrapable cofactor is selected to minimize diffusionof the entrappable cofactor throughout the first transport material. 5.The sensor to measure the presence of an analyte as described in claim 4further including: an insulation layer covering the working conductor,the insulation layer having at least one aperture that exposes theelectrode reactive surface.
 6. The sensor to measure the presence of ananalyte as described in claim 5, wherein the reactive chemistry isdisposed within the aperture within the insulation layer.
 7. The sensorto measure the presence of an analyte as described in claim 6, whereinthe reactive chemistry covers a portion of the electrode reactivesurface.
 8. The sensor to measure the presence of an analyte asdescribed in claim 6, wherein the reactive chemistry covers an entiretyof the electrode reactive surface.
 9. The sensor to measure the presenceof an analyte as described in claim 8, wherein the reactive chemistryfills the aperture.
 10. The sensor to measure the presence of an analyteas described in claim 9, wherein the reactive chemistry further covers aportion of the insulation.
 11. The sensor to measure the presence of ananalyte as described in claim 6, wherein the second transport materialis applied over the insulation layer and the reactive chemistry.
 12. Thesensor to measure the presence of an analyte as described in claim 11,wherein the tunable diffusivity of the entrappable cofactor and theporosity of the first transport material are selected to minimizediffusion of the entrappable cofactor into the second transportmaterial.
 13. The sensor to measure the presence of an analyte asdescribed in claim 6, wherein the reactive chemistry further includes aconductive polymer.
 14. The sensor to measure the presence of an analyteas described in claim 1, wherein the anchor molecule has a molecularmass between 500 and 50,000 daltons.
 15. The sensor to measure thepresence of an analyte as described in claim 1, wherein a preferredelectrical charge associated with the anchor molecule establishes thetunable diffusivity of the entrappable cofactor.
 16. The sensor tomeasure the presence of an analyte as described in claim 1, wherein theanchor molecule has a molecular mass between 500 and 50,000 daltons andimparts a preferred electrical charge to the entrappable cofactor. 17.The sensor to measure the presence of an analyte as described in claim15, wherein the first transport material has an electrical charge beingopposite the preferred electrical charge of the anchor molecule.
 18. Thesensor to measure the presence of an analyte as described in claim 16,wherein the first transport material has a porosity that encumbersdiffusion of molecules having a molecular mass greater than 500 daltonsand an electrical change being opposite the preferred electrical chargeof the anchor molecule.
 19. The sensor to measure the presence of ananalyte as described in claim 9, wherein the second transport materialis applied over the reactive chemistry and a portion of the insulation.20. The sensor to measure the presence of an analyte as described inclaim 9, wherein the second transport material covers an entirety of theinsulation layer and the reactive chemistry.